At the end of its lifespan the human erythrocyte is recognized, sequestered and digested by phagocytic cells of the reticuloendothelial system. The proposed work is attempting to establish which of the physicochemical changes that occur during aging of RBC in vivo is primarily responsible for triggering the recognition system. In order to accomplish this aim populations of RBC established to be senescent are required. Consequently the operational efficiency of a number of cell density fractionation procedures are now under evaluation. The methods under evaluation include the centrifugal technique of Murphy, percoll, stractan and albumin density gradient separations. The most effective technique appears to be that of Murphy which has a low inefficiency number of about 0.2. Recent work has provided evidence for the accumulation of membrane-bound IgG as the RBC ages in vivo. Attempts are now being made to establish the best conditions for detecting the amount of IgG on the surface of an RBC including the use of a fluorescence activated cell sorter, a radioimmunoassay technique using I125-labeled anti-human IgG and an enzyme linked immunosorbent assay (ELISA). Specific membrane modification techniques will be employed to produce chemically defined alterations in membrane properties which are found to be critical or relevant to the in vivo recognition and elimination process for senescent red cells. In vitro tests including erythrophagocytosis will be refined for monitoring membrane alterations as reflected by variation in the biological behavior of the cells as assessed by red cell-macrophage interaction.